A disturbing consequence of the use, and over-use, of beta-lactam antibiotics (e.g., penicillins and cephalosporins) has been the development and spread of beta-lactamases. Beta-lactamases are enzymes that open the beta-lactam ring of penicillins, cephalosporins, and related compounds, to inactivate the antibiotic. The production of beta-lactamases is an important mechanism of resistance to beta-lactam antibiotics among Gram-negative bacteria.
Expanded-spectrum cephalosporins have been specifically designed to resist degradation by the older broad-spectrum beta-lactamases such as TEM-1, 2, and SHV-1. Microbial response to the expanded-spectrum cephalosporins has been the production of mutant forms of the older beta-lactamases called extended-spectrum beta-lactamases (ESBLs). Although ESBL-producing Enterobacteriaceae were first reported in Europe in 1983 and 1984, ESBLs have now been found in organisms of diverse genera recovered from patients in all continents except Antarctica. The occurrence of ESBL-producing organisms varies widely with some types more prevalent in Europe (TEM-3), others more prevalent in the United States (TEM-10, TEM-12 and TEM-26), while others appear worldwide (SHV-2 and SHV-5). These enzymes are capable of hydrolyzing the newer cephalosporins and aztreonam. Studies with biochemical and molecular techniques indicate that many ESBLs are derivatives of older TEM-1, TEM-2, or SHV-1 beta-lactamases, some differing from the parent enzyme by one to seven amino acid substitutions.
In addition, resistance in Klebsiella pneumoniae and Escherichia coli to cephamycins and inhibitor compounds such as clavalante have also arisen via acquisition of plasmids containing the chromosomally derived AmpC beta-lactamase, most commonly originating from Enterobacter cloacae, Citrobacter freundii, Hafnia alvei, and Morganella morganii. 
It is of particular concern that genes encoding the beta-lactamases are often located on large plasmids that also contain genes for resistance to other antibiotic classes including aminoglycosides, tetracycline, sulfonamides, trimethoprim, and chloramphenicol. Furthermore there is an increasing tendency for bacteria to produce multiple beta-lactamases. These developments, which occur over a wide range of Gram-negative genera, represent a recent evolutionary development in which common Gram-negative bacteria are availing themselves of increasingly complex repertoires of antibiotic resistance mechanisms. Clinically, this increases the difficulty of identifying effective therapies for infected patients.
Organisms overexpressing AmpC beta-lactamases are a major clinical concern because these organisms are usually resistant to all the beta-lactam drugs except the dipolar ionic methoxyiminocephalosporins such as cefepime and cefpirome and the carbapenems. However, recently an Enterobacter cloacae clinical isolate associated high-level resistance to cefepime and cefpirome with overexpression of and a deletion within the ampC structural gene was reported. Barnaud et al., FEMS Microbiology Letters, 195:185-190 (2001).
Overexpression of AmpC beta-lactamases can occur in two ways, the deregulation of the chromosomal gene expressing the AmpC beta-lactamase or the acquisition by gram-negative organisms of a transferable ampC gene either on a plasmid or other transferable element. The latter have commonly been called plasmid-mediated AmpC beta-lactamases.
The ability to identify the difference between constitutive overexpression of AmpC beta-lactamase from the chromosome or a plasmid is important for hospital epidemiology. Organisms with inducible chromosomal ampC beta-lactamase genes include E. cloacae, E. aerogenes, Citrobacterfreundii, Morganella morganii, Hafnia alvei, Serratia marcescens, and indole positive Proteus spp. Strains of these organisms that overexpress the chromosomal genes are collectively called derepressed mutants. Therefore, by identifying the organism the laboratory can identify the ability of that organism to overexpress the AmpC beta-lactamase. Escherichia coli strains can also overexpress their chromosomal ampC beta-lactamase gene and are termed hyperproducing E. coli. Plasmid-mediated ampC genes are derived from the chromosomal ampC gene of several members of the family Enterobacteriaceae, such as E. cloacae, C. freundii, and others. But not all members of the family Enterobacteriaceae encode a gene for AmpC beta-lactamases or are the origin of plasmid-mediated genes, such as K. pneumoniae or E. coli, respectively. Therefore, the distinction between a plasmid-mediated AmpC beta-lactamase and the endogenous enzyme is difficult to determine in both hyperproducing E. coli strains and organisms with inducible chromosomal AmpC enzymes. This distinction, however, is critical for hospital infection control. Plasmid-mediated genes whether they are extended-spectrum beta-lactamases (ESBLs) or AmpC enzymes can spread rapidly to members of the same species or organisms of different genera. In addition, multiple beta-lactamases within one organism, such as multiple ESBLs or a combination of ESBLs and AmpC enzymes can make phenotypic identification of the AmpC enzyme difficult. Unfortunately, for these reasons, the detection of AmpC, particularly plasmid-mediated AmpC, beta-lactamase resistance goes undetected in most clinical laboratories.
The ability to distinguish between different types of ampC beta-lactamase nucleic acid in a clinical sample is useful for such epidemiological purposes as identifying how particular bacteria of interest have spread, thus aiding in infection control. It is also useful for identifying the proper antibiotic treatment for a patient. Thus, there is a need for techniques that can quickly and accurately identify the particular types of beta-lactamases that may be present in a clinical isolate or sample, for example. This could have significant implications in the choice of antibiotic necessary to treat a bacterial infection.